Straw for the preservation of a predetermined dose of liquid-based substance, in particular diluted animal semen; and system comprising such a straw and a dilution extender to give such a substance

ABSTRACT

The straw comprises a tube ( 11 ) extending between a first end and a second end and comprises a gas-permeable, liquid-tight plug, said plug being arranged in the tube ( 11 ) in the vicinity of the first end of same and extending between a first end turned towards the first end of the tube ( 11 ) and a second end turned towards the second end of the tube ( 11 ). Said plug comprises an element ( 14 ) impregnated with multivalent cations ( 22 ) at least in the vicinity of a first end turned towards the second end of the tube ( 11 ). The system comprises the straw and a liquid medium provided to produce, by mixing, in predefined conditions, the liquid substance ( 21 ). The medium contains an alginate ( 23 ) in solution.

The invention generally concerns the preservation of a predetermineddose of liquid-based substance containing biological material. Such asubstance is for example diluted animal semen.

More particularly, the invention concerns the straws for implementingsuch preservation.

It is known that these straws are conventionally formed by a thin tube,having for example an inner diameter of 1.6 or 2.5 mm, and by a stopperinserted in the thin tube.

In the filled state, the stopper is arranged close to a first end of thetube and the dose of liquid-based substance is arranged in the strawbetween the stopper and the second end of the tube.

In order to fill the straw, the first end of the tube, close to thestopper, is placed in communication with a vacuum source, while thesecond end is placed in communication with a vessel containing thesubstance to be introduced into the straw. The air initially containedbetween the stopper and the second end is sucked through the stopperwhile the substance moves forward into the tube until it reaches thestopper, which it cannot pass because the stopper becomes liquid-tight.

If necessary, after filling, the straw is welded close to one or both ofits ends and is stored cold.

In order to empty the straw, if necessary after cutting the welded endportions and thawing, a rod is inserted into the tube via the endclosest to the stopper, until it bears against the stopper. Using thisrod, the stopper is made to slide in the manner of a piston towards theend furthest from the stopper, so that the dose of substance initiallycontained in the straw is expelled through that end.

Straw stoppers are generally of the three-part type originally describedin French patent 995.878, corresponding to British patent 669,265, i.e.formed by two plugs made from a fibrous substance enclosing a powderwhich, on contact with a liquid, is transformed into an impermeablepaste or gel adhering to the wall of the tube so that the stopper isliquid-tight.

European patent application EP 0 873 726 proposes that the stopper beformed by a single-piece cylinder of hydrophobic microporous material.

French patent applications 2 771 285 and 2 784 572, to which Americanpatent application US 2001/0014376 and American patent U.S. Pat. No.6,416,611 correspond, propose that the stopper be constituted by a stiffinsert perforated by a substantially coaxial orifice and a hydrophobicmicroporous membrane combined with the insert in order to seal theinsert orifice on the inside.

French patent applications 2 824 255 and 2 824 256, to which Americanpatent applications US 2002/0183653 and US 2002/0188222 correspond,propose to add to the stopper, besides the powder and fibres,non-absorbent elements, namely a core made from thermoplastic material,covered with a sleeve made from braided threads, and non-absorbentmaterial in dispersed form, in the powder.

The invention is directed to limiting the loss, in the stopper of such astraw, of the product of interest contained in the liquid-basedsubstance, for example spermatozoids if the substance is diluted semen.

To that end the invention provides a straw for the preservation of apredetermined dose of liquid-based substance containing biologicalmaterial, comprising a tube extending between a first end and a secondend and comprising a liquid-tight, gas-permeable stopper, which stopperis disposed in the tube close to its first end and extends between afirst end facing towards the first end of the tube and a second endfacing towards the second end of the tube; characterized in that saidstopper comprises a member impregnated with multivalent cations at leastin the neighborhood of a first end facing towards the second end of thetube.

It is known that in an aqueous medium, multivalent cations have thecapacity to cross-link alginates.

Therefore, if the liquid-based substance contains an alginate, incontact with the second end of the stopper of the straw, the substancethickens and forms a matrix or network which cannot be traversed by theproduct of interest, for example spermatozoids, which cannot thereforepenetrate into the stopper where it would be lost.

The straw according to the invention thus makes it possible to limit theloss of the product of interest in the stopper of the straw.

It will be noted that it is possible to add an alginate to aliquid-based substance in a proportion such that the alginate can reactwith the multivalent cations as has just been indicated, without thisaddition influencing the viscosity of the liquid-based substance or elseinfluencing that viscosity minimally. Therefore, this addition haslittle or no influence on the product of interest contained in thesubstance, for example spermatozoids. Similarly, this addition haslittle or no influence on the cooperation of the liquid-based substancewith the straw, apart from the above-mentioned cross-linking of thealginate.

It will be noted that the impregnation of the stopper of the straw atleast in the vicinity of the second end by multivalent cations is arelatively simple operation to carry out. The straw according to theinvention is thus simple, convenient and economic to produce.

According to advantageous features:

-   -   said multivalent cations are divalent cations;    -   said divalent cations comprise barium Ba⁺⁺cations;    -   said divalent cations comprise calcium Ca⁺⁺cations;    -   said stopper comprises two plugs made from a fibrous substance        enclosing a sealing agent formed by a powder which, on contact        with a liquid, transforms into an impermeable paste or gel        adhering to the wall of the tube so that the stopper is        liquid-tight;    -   in the stopper of the straw, the plug situated towards the        second end of the tube forms said impregnated member, said first        end of the impregnated member forming said second end of the        stopper;    -   said stopper comprises a gas-permeable, liquid permeable barrier        plug extending between a first end facing towards the first end        of the tube and a second end facing towards the second end of        the tube, the first end of the impregnated member and the second        end of the barrier plug being disposed against each other;    -   the barrier plug is hydrophobic; and/or    -   said impregnated member is a plug entirely impregnated with        divalent cations.

The invention is also directed to a system for the preservation ofbiological material, characterized in that it comprises:

-   -   a straw as set out above for the preservation of a predetermined        dose of liquid-based substance containing biological material;        and    -   a liquid dilution extender to give by mixing, in predetermined        conditions, said liquid-based substance containing biological        material; which extender contains an alginate in solution.

The liquid-based substance obtained with this liquid medium thuscontains an alginate and is suitable for being used with a straw as setforth above.

Advantageously, said liquid-based substance is diluted animal semen andsaid alginate is in a concentration comprised between 0.1 and 6 g/l; andstill more advantageously in a concentration comprised between 2.5 and 5g/l.

The disclosure of the invention will now be continued with the detaileddescription of embodiments, given below by way of illustrative andnon-limiting example, with reference to the accompanying drawings, inwhich:

FIG. 1 is a diagrammatic view in longitudinal cross-section of a strawaccording to the invention, in the empty state;

FIG. 2 is a view similar to FIG. 1 but showing the straw in the filledstate;

FIG. 3 is an enlargement of the part of FIG. 2 situated around the endof the stopper which can be seen to the right;

FIG. 4 diagrammatically illustrates a bottle containing a liquiddilution extender, a container containing pure animal semen and acontainer containing diluted animal semen obtained by dilution of thatsemen with that extender; and

FIG. 5 is a partial view of a variant of the straw, in which the stopperfurther comprises a barrier plug.

The straw 10 illustrated in FIG. 1 comprises a tube 11 and a stopper 12.

The tube 11 is conventionally made from extruded plastic material, heretransparent, with an inside diameter for example of 1.6 or 2.6 mm and alength of the order of 133 mm.

The stopper 12 is of the three-part type, i.e. formed by two plugs 13and 14 made from a fibrous substance, for example of braided threads,enclosing a sealing agent 20 formed by a powder (FIG. 1) which, oncontact with a liquid, is capable of transforming into an impermeablepaste or gel 15′ (FIG. 2) adhering to the wall of the tube 11 so thatthe stopper 12 is liquid-tight.

In the initial state, shown in FIG. 1, the stopper 12 is disposed in theneighborhood of the end 16 of the tube 11 and it is provided that in thefilled state, the dose of liquid-based substance which must be preservedin the straw 10 is disposed between the stopper 12 and the end 17 of thetube 11 that is the furthest from the stopper 12.

In order to fill the straw 10, the end 16 is placed in communicationwith a vacuum source while the end 17 is placed in communication with avessel containing the substance to be introduced into the straw.

The air initially contained between the stopper 12 and the end 17 issucked through the stopper 12 while the substance 21 (FIG. 2) movesforward in the tube 11 until it encounters the stopper 12, by the end 18thereof that faces towards the end 17 of the tube 11, that is to say theend of the stopper 12 that can be seen on the right in FIGS. 1 and 2.

The straw 10 is then in the filed state shown in FIG. 2.

If necessary, after filing, the straw is welded in the neighborhood ofone or both of its ends 16 and 17 and is placed in cold storage.

To empty the straw 10, if necessary after cutting the welded endportions and thawing, there is inserted into the tube 11 a rod whichcomes to bear on the end 19 of the stopper 12 (which end is situated onthe opposite side to the end 18).

Using this rod, the stopper 12 is made to slide in the manner of apiston towards the end 17 or the end which corresponds after cutting thewelded portion, which causes the expulsion of the dose of substance 21which had been introduced into the straw.

With reference to FIG. 3, a description will now be given in more detailof the plug 14 of the stopper 12 of the straw 10 and of the zonesituated in the neighborhood of its end 18 when the straw 10 is in thefilled state shown in FIG. 2.

The plug 14 is impregnated by divalent cations 22, here cations ofbarium, Ba⁺⁺.

The substance 21 here is diluted animal semen which contains analginate.

It is known that in an aqueous medium, divalent cations such as thecations 22, have the capacity to cross-link alginates such as thealginate 23.

Thus, when the substance 21 came into contact with the end 18 of thestopper 12, the substance 21 thickened and formed a matrix or network24.

The matrix or network 24 is such that the spermatozoids 25 contained inthe substance 21 cannot traverse the matrix or network 24.

Therefore, the spermatozoids 25 cannot penetrate into the stopper 12,where they would be lost.

Here, the plug 14 is entirely impregnated with divalent cations 22, forreasons of convenience of manufacture,

For example, the plug 14 is manufactured from a section of a braidedwick which had been soaked in a saturated solution of barium salt thenremoved from the bath and placed to dry before being cut up intosections one of which forms the plug 14.

As a variant, only the region near the end 18 is impregnated withdivalent cations 22.

FIG. 4 shows a bottle 26 containing a dilution extender 27, a container28 containing pure animal semen 29 and the same container 28 containingdiluted animal semen, forming the substance 21, obtained by dilution ofthe pure animal semen 29 with the dilution extender 27.

It is clearly understood that the dilution extender 27 has not yet beenmixed with pure semen, and is thus distinct from diluted semen.

The dilution extender 27 is for example that commercialized by theapplicant under the name OptiXcell.

The dilution of the pure semen 29 by the extender 27 is carried out inpredetermined conditions, in particular as regards the dilution ratio,that is to say the ratio between the total volume of the diluted semen21 and the volume of the pure semen 29.

The dilution extender 27 contains the alginate 23, which has beenselected to cross-link in contact with divalent cations 22 in order toform the matrix or network 24, which is thus a thickened zone of thediluted semen 21.

A range of concentration which is particularly suitable is the rangefrom 0.1 g to 6 g of alginate per liter of dilution extender 27. Aparticularly advantageous range extends between 2.5 g/l and 5 g/l.

For example, with a concentration of alginate of the order of 2.5 g/lthe proportion of liquid-based substance lost in the stopper 12 is from3 to 4%; and with a concentration of the order of 5 g/l, the proportionof liquid-based substance lost in the stopper such as 12 is of the orderof 1 à 3%.

It will be noted that this proportion of liquid lost in the stopper isdetermined by calculating the difference between the quantity ofliquid-based substance introduced into the straw until the stopper 12becomes fluid-tight and the quantity of liquid which the straw canreturn by sliding the stopper 12 as explained above.

It will be noted that in FIGS. 2 and 3, the matrix or network 24 isshown only in the vicinity of the end 18 of the stopper 12.

According to the concentration of the extender 27 in alginate, insteadof having a localized presence of the matrix or network 24, asillustrated in FIGS. 2 and 3, the matrix or network may extend to arelatively great distance from the end 18 of the stopper 12.

The variant of the straw 10 shown in FIG. 5 is similar to the straw 10shown in FIGS. 1 to 3 apart from the fact that the stopper 12 furthercomprises a barrier plug 30 towards the end 17 of the tube 11.

The barrier plug 30 extends between an end 31 facing towards the end 17of the tube 11 and an end 32 facing towards the end 16 of the tube 11.

In the straw 10 shown in FIGS. 1 to 3 the end 18 of the stopper 12facing towards the end 17 of the tube 11 forms part of the plug 14. Inthe variant of the straw 10 shown in FIG. 5, the end 18 forms part ofthe barrier plug 30 and the plug 14 has, on the side facing towards theend 17, an end 33 distinct from the end 18.

The end 33 of the plug 14 and the end 32 of the barrier plug 30 arearranged against each other.

The barrier plug 30 is fibrous. It is gas-permeable andliquid-permeable.

The straw 10 shown in FIG. 5 is used in the same way as the straw 10shown in FIGS. 1 to 3.

On filling, the substance 21 traverses the barrier plug 30 andencounters the plug 14 by its end 33 that faces towards the end 17 ofthe tube 11. In the same way as for the straw 10 shown in FIGS. 1 to 3,the substance 21 thickens and forms the matrix or network 24.

The barrier plug 30 enables the matrix or network 24 to remain in thestopper 12: the barrier plug 30 prevents the passage of the matrix ornetwork 24 to the substance 21.

The barrier plug 30 is here a braid formed by threads arranged in a coreand a cover surrounding the core.

Each thread is hydrophobic. Therefore, the barrier plug 30 ishydrophobic, and thus has a water-repellant effect.

This repellant effect does not prevent the substance 21 from passingthrough the barrier plug 30 and reaching the impregnated plug 14, sincein practice the substance 21 comes up against the stopper 12 with acertain speed.

During the passage of the substance 21 into the barrier plug 30, thethreads forming it do not absorb liquid; and after the passage of thesubstance 21 is blocked by the stopper 12, the barrier plug 30 does notkeep the liquid situated in its interstices but returns it into the doseof liquid substance situated between the end 31 of the barrier plug 30and the end 17 of the tube 11.

As a result, there is no consumption, or very reduced consumption, ofliquid substance by the barrier plug 30.

In a variant not illustrated, in particular when the concentration ofalginate in the dilution extender 27 is relatively high, the three-partstopper 12 is replaced by a stopper formed only by a plug such as theplug 14.

In a variant not illustrated, the stopper 12 is arranged differently,for example made from a one-piece cylinder of hydrophobic microporousmaterial such as described in European patent application EP 0 873 726.

In variants not illustrated, the divalent cations 22 formed by bariumions Ba⁺⁺are replaced by other divalent cations, for example calciumCa⁺⁺or magnesium Mg⁺⁺cations.

In variants not illustrated, the divalent cations 22 are replaced bymultivalent cations other than divalent ones, for example trivalentcations such aluminum Al⁺⁺⁺or chromium Cr⁺⁺⁺cations.

In other variants not illustrated, the liquid-based substance containingthe biological material is different from diluted animal semen, forexample a preservation medium containing embryos.

Numerous other variants are possible according to circumstances, and inthis connection it is to be noted that the invention is not limited tothe examples described and shown.

1. A straw for the preservation of a predetermined dose of liquid-basedsubstance containing biological material, comprising a tube (11)extending between a first end (16) and a second end (17) and comprisinga liquid-tight, gas-permeable stopper (12), which stopper (12) isdisposed in the tube (11) close to its first end (16) and extendsbetween a first end (19) facing towards the first end (16) of the tube(11) and a second end (18) facing towards the second end (17) of thetube (11); characterized in that said stopper (12) comprises a member(14) impregnated with multivalent cations (22) at least in theneighborhood of a first end (18; 33) facing towards the second end (17)of the tube (11).
 2. A straw according to claim 1, characterized in thatsaid multivalent cations are divalent cations.
 3. A straw according toclaim 2, characterized in that said divalent cations comprise bariumBa⁺⁺cations (22).
 4. A straw according to claim 2, characterized in thatsaid divalent cations comprise calcium Ca⁺⁺cations.
 5. A straw accordingto claim 1, characterized in that said stopper (12) comprises two plugs(13, 14) made from a fibrous substance enclosing a sealing agent (20)formed by a powder (15) which, on contact with a liquid, transforms intoan impermeable paste or gel (15′) adhering to the wall of the tube sothat the stopper is liquid-tight.
 6. A straw according to claim 5,characterized in that, in the stopper (12) of the straw (10), the plug(14) situated towards the second end (17) of the tube (11) forms saidimpregnated member, said first end (18) of the impregnated memberforming said second end of the stopper.
 7. A straw according to claim 1,characterized in that said stopper (12) comprises a gas-permeable,liquid permeable barrier plug (30) extending between a first end (31)facing towards the first end (17) of the tube (11) and a second end (32)facing towards the second end (16) of the tube (11), the first end (33)of the impregnated member (14) and the second end (32) of the barrierplug (30) being disposed against each other.
 8. A straw according toclaim 7, characterized in that the barrier plug (30) is hydrophobic. 9.A straw according to claim 1, characterized in that said impregnatedmember is a plug (14) entirely impregnated with divalent cations (22).10. A system for the preservation of biological material, characterizedin that it comprises a straw (10) according to claim 1, for thepreservation of a predetermined dose of liquid-based substancecontaining biological material; and a liquid dilution extender (27) togive by mixing, in predetermined conditions, said liquid-based substancecontaining biological material (21); which extender contains an alginate(23) in solution.
 11. A system according to claim 10, characterized inthat said liquid-based substance is diluted animal semen and saidalginate is in a concentration comprised between 0.1 and 6 g/l.
 12. Asystem according to claim 11, characterized in that said alginate is ina concentration comprised between 2.5 and 5 g/l.